Mycobacterial Diseases
Addo et al., Mycobact Dis 2016, 6:5
DOI: 10.4172/2161-1068.1000229
Research Article
OMICS International
Prevalence of Tuberculosis Infection in a Cohort of Cattle that Enters the Food
Chain in Accra, Ghana using Bovigam
Kennedy Kwasi Addo*, Vida Yirenkyiwaa Adjei, Gloria Ivy Mensah and Dolly Jackson-Sillah
Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
*Corresponding author: Kennedy Kwasi Addo, Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana; Tel:
+23324333486; Fax: +233 302502182; E-mail: [email protected]
Rec date: September 27, 2016; Acc date: December 06, 2016; Pub date: December 12, 2016
Copyright: © 2016 Addo KK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Tuberculosis (TB) continues to be an important public health problem worldwide. Currently there are 286 TB
cases per every 100,000 people in Ghana. This figure is three times higher than the TB burden estimated by the
World Health Organization. The numerical contribution of bovine TB to the general TB burden is unknown.
Herdsmen, livestock workers, and veterinarians and the general public are at high risk of contracting bovine TB.
Zoonotic TB caused by Mycobacterium bovis is present in animals in most developing countries including Ghana.
Unfortunately, activities to check and control cattle with TB infection from entering the food chain are often
inadequate or unavailable. This study therefore aims to determine the prevalence of TB infection in a cohort of cattle
that enters the food chain in Accra, Ghana. A cross sectional study involving five major abattoirs was conducted in
the Greater Accra region between the periods of September 2012-June 2013. After routine inspection of live cattle
by veterinary officials, 10 mL of blood was drawn from 94 cattle before slaughter and tested for TB infection using
BOVIGAM. Six (6.4%) of the 94 cattle screened, were positive for TB infection. All except one abattoir had at least
one cattle testing positive. Although the study recorded a lower prevalence of 6.4%, all animals tested were deemed
fit for slaughter by veterinary officials and was to enter the food chain. The low sensitivity of routine abattoir
inspection for infected organs and negative results of post-mortem examination reinforces the need for more
sensitive screening tool such as BOVIGAM.
Keywords: Bovine Tuberculosis; BOVIGAM; Gamma interferon (γIFN); Abattoir; Ghana
Introduction
Tuberculosis (TB) is caused by a group of bacteria collectively
known as the Mycobacterium tuberculosis complex [1]. The
commonest strain of Mycobacterium found among TB patients in
Ghana is the M. tuberculosis followed by Mycobacterium africanum
and Mycobacterium bovis [2]. M. bovis is virulent for cattle but can
infect other animals and humans causing disease and pathology
similar to M. tuberculosis, which is naturally pathogenic for man [3,4].
It has been established that 3% of pulmonary TB in Accra, Ghana is
caused by M. bovis [2] which raises concern about possible aerosol
transmission between cattle and human population or within the
human population. Despite rigorous control efforts, the current global
estimates indicate that 1/3 of the world’s population has TB infection
and 5-10% of these individuals if HIV negative will develop active TB
during their lifetime, contributing to a global annual incidence of
approximately 9.2 million cases [5]. A study in the Ho district of the
Volta Region of Ghana revealed a prevalence rate of 3.1% bovine TB
infection in cattle and 5.9% within a cluster [6] whilst and others have
also indicated transmission from humans to animals and vice versa [7].
Abattoirs provide ideal settings for screening of cattle because they are
the last point for inspection before slaughter and it is critical that TB is
detected at this time to prevent TB-infected cattle in our food chain.
Over the years, in vivo intradermal comparative tuberculin skin test
has been used as standard test for diagnosis of TB in cattle worldwide,
in spite of lacking the sensitivity and specificity [8]. Monitoring bovine
TB in cattle by bacteriological assay is not feasible, costly, time
Mycobact Dis, an open access journal
ISSN:2161-1068
consuming and most laboratories are ill equipped [9]. In recent years,
gamma interferon (γ-IFN) assay has been used for detection of bovine
TB in that it detects the cytokine γ-interferon which is predominantly
released by T-cells after in vitro stimulation with bovine Purified
Protein Derivative (BvPPD) and avian Purified Protein Derivative
(AvPPD) [10,11]. BOVIGAM® is an example of gamma interferon (γIFN) assay used for the diagnosis of bovine TB infection in cattle.
Animals infected with M. bovis can be identified by measuring the
cytokine interferon gamma (IFN-γ) against tuberculin, an antigen
used to aid in the diagnosis of TB infection. Tuberculin Purified
Protein Derivatives (PPD) antigens are presented to lymphocytes in
whole blood cultures and the production of IFN-γ from the stimulated
T cells is detected using a monoclonal antibody-based sandwich
enzyme immunoassay (EIA). Lymphocytes from uninfected cattle do
not produce IFN-γ to tuberculin PPD antigens and hence IFN-γ
detection correlates with infection.
This study aimed to determine the prevalence of TB infection in a
cohort of cattle that enter the food chain in Accra, Ghana using
Bovigam.
Material and Method
Study design
This was a cross sectional study involving five major abattoirs
namely: Tema abattoir (GIHOC), Madina abattoir, University of
Ghana farms (Legon Abattoir), Accra abattoir and Amasaman abattoir
in the Greater Accra region of Ghana. Abattoir practices, including the
extent of examination of live animals (ante mortem) and carcasses
Volume 6 • Issue 5 • 1000229
Citation:
Addo kk, Adjei VY, Mensah GI, Jackson-Sillah D (2016) Prevalence of Tuberculosis Infection in a Cohort of Cattle that Enters the Food
Chain in Accra, Ghana using Bovigam. Mycobact Dis 6: 229. doi:10.4172/2161-1068.1000229
Page 2 of 3
(post mortem) and what is done when there is a visible lesion was
observed and recorded by a trained veterinarian.
Sample size
Ninety-four cattle slaughtered for consumption were involved.
Because slaughtering of cattle was not regular in the selected abattoirs
only 94 animals were available for sampling during the period of
sample collection.
Sample collection
Sample collection was done shortly after the animals had been
inspected and declared fit for slaughter. 10 mL of blood was drawn
from the jugular vein for Bovigam test between September 2012 and
June 2013. Handling of blood from cattle and the laboratory analysis
was conducted at the Noguchi Memorial Institute for Medical
Research.
Bovigam test
Stage one-whole blood culture
A minimum volume of 5 mL of blood from each animal was
collected into a heparinised blood collection tube and mixed evenly.
Three 1.5 mL aliquots of heparinised blood from each animal were
aseptically dispensed into wells of 24-well tissue culture trays. The
wells were labeled; Phosphate Buffered Saline (PBS), AvPPD and
BvPPD. 100 µL of PBS, (NIL Antigen control), avian PPD and bovine
PPD were aseptically added to the appropriate wells containing
previously dispensed blood. The culture tray was swirled ten times
both clockwise and counter clockwise on a flat smooth surface to mix
for 1 minute. The culture tray containing blood and antigens was
incubated for 16-24 h at 37°C in a humidified atmosphere. 500 µL of
plasma was harvested into sterile Eppendorf tubes and stored at -20°C
prior to ELISA.
Stage two - Bovine IFN-γ EIA
All test plasmas and reagents except the Conjugate 100X
concentrate were brought to room temperature (22+/-3°C) before use.
Freeze dried component was reconstituted based on the manufacturer’s
instruction.
50 µL of Green Diluent was added to the required wells. 50 µL of test
and control samples were also added to the appropriate wells
containing the Green Diluent. The control samples were added last.
The plates were then placed on a microplate shaker to mix the content
thoroughly for 1 min, after which they were incubated at room
temperature on the microplate shaker at a setting of 600 rpm for 1 h.
The content was poured out and washed six times with freshly
prepared wash buffer. After the six washes, plates were placed face
down on clean filter paper and allowed to drain and flick several times
over absorbent paper to remove excess wash buffer as possible. 100 µL
of freshly prepared conjugate reagent was added to the wells and
incubated on a microplate shaker set at 600 rpm for 1 h (conjugate
reagent was prepared based on manufacturer’s instruction). The
content was again poured out and washed six times with wash buffer.
100 µL of freshly prepared enzyme substrate solution was added to the
wells and mixed thoroughly in a microplate shaker. The plates were
covered and incubated on the microplate shaker set at 600 rpm in a
dark area for 30 min. 50 µL of the enzyme stopping solution was added
Mycobact Dis, an open access journal
ISSN:2161-1068
to each well and agitated gently, while care was taken not to transfer
chromogen from well to well. Absorbance of each well was measured
within 5 min of terminating the reaction using a microplate reader
fitted with 450 nm filter. The absorbance value was used to calculate
the results. Blood plasma collected from cattle having an OD value
greater than 0.100 above that of avian PPD and nil (PBS) antigen,
indicates the presence of M. bovis infection.
Results
Blood samples were collected from cattle declared as "good for
slaughter" upon clinical examination by veterinary officers. Blood
samples were collected from 94 cattle from 5 abattoirs, namely Legon
(6), Accra (26), Madina (24), Amasaman (20) and GIHOC (18).
Of the 94 cattle screened for TB infection using the Bovigam test, 6
(6.4%) were positive for TB infection. All except Legon abattoir had at
least one cattle testing positive (Table 1). We could not find an
association between Bovigam result and abattoir of cattle (χ2 test: p=0.
841).
Abattoir
No. of cattle tested
Bovigam (IFN-ɣ assay)
Positive
Negative
Legon
6
0
6
Accra
26
2
24
Madina
24
1
23
Amasaman
20
1
19
GIHOC
18
2
16
Total
94
6
88
Table1: Bovigam results by facility.
Discussion
More than a decade ago, the TB prevalence in cattle around the
Accra plains was estimated at 11-19 % using the intradermal
tuberculin (ITT) skin test [12]. This present study using the Bovigam
test, however, recorded a much, lower prevalence of 6.4%. This figure
does not suggest a reduction in TB cases in cattle, but is rather a
reflection of the test used which is more specific than the ITT used in
the previous study. This assertion is supported by several studies that
have reported that the ITT is less sensitive and specific than the
Bovigam [10,13-15].
In our study, all the animals tested with the Bovigam, had been
deemed fit for slaughter, after routine examination by veterinary
officials. The examination involved evaluation of the general body
condition scored as 1 "good'', 2 ''bad'' and 3 ''very bad'' as well as
inspection of inguinal lymph node and prescapular lymph node judged
as normal or enlarged. In spite of the animals being declared ''free of
TB infection'' by clinical inspection, 6 out of the 94 were positive for
TB infection by the Bovigam test. The TB infection could have been
missed during the clinical inspection because the infection was in the
early stages or latent.
At the early stages of TB infection or latent, the pathological
changes caused by the bacilli are not yet profound enough to be
detectable during routine abattoir inspection. From this period till the
Volume 6 • Issue 5 • 1000229
Citation:
Addo kk, Adjei VY, Mensah GI, Jackson-Sillah D (2016) Prevalence of Tuberculosis Infection in a Cohort of Cattle that Enters the Food
Chain in Accra, Ghana using Bovigam. Mycobact Dis 6: 229. doi:10.4172/2161-1068.1000229
Page 3 of 3
development of detectable signs of bovine TB, cellular immune
responses will however be detectable earlier than the pathological
changes caused by the disease (e.g., visible lesions), and before the
bacterial loads exceed the numbers necessary for the detection of M.
bovis from tissue samples by culture[16]. This is one major advantage
of Bovigam over other tests that rely on detection of bacillus or signs of
active infection. Bovigam essentially detects infection rather than
disease so can be used as a screening tool in large herds to prevent the
spread of infection by detection and subsequent quarantine of infected
cattle early before visible signs of TB are seen.
Compared to other studies in Turkey [17] and Cameroon[18] which
recorded a TB prevalence of 49.2% and 60%, respectively, in cattle
bound for slaughter, the prevalence of 6.4% recorded in our study is on
the lower side.
2.
3.
4.
5.
6.
7.
The low prevalence notwithstanding, it is still a cause for concern as
these cattle had been adjudged to be fit for slaughter and was to enter
the food chain. Our study also highlights the low sensitivity of routine
abattoir inspections for infected organs and reinforces the need for
more sensitive screening tools such as the Bovigam. A recent study in
Ethiopia has revealed that the probability of missing an animal with a
TB lesion during routine abattoir inspection is 95.24% [19].
8.
Additionally, in many of such cases even post mortem investigation
does not yield any positive results, because due to the low bacteria load
or latent stage of the disease, organs would appear normal.
Consumption of undercooked meat from such cattle could potentially
lead to infection.
11.
Also, considering the fact that many animals are slaughtered outside
the abattoir system where there are no clinical examinations, the
prevalence of TB in cattle entering the food change will be much
higher.
13.
9.
10.
12.
14.
Acknowledgements
This work was funded by the University of Ghana Research Fund
Number: URF/5/ILG-006/2011-2012 and supported partially by the
consortium Afrique One “Ecosystem and Population Health:
"Expanding Frontiers in Health” funded by the Wellcome Trust
(WT087535MA) and Afrique One – ASPIRE (107753/A/15/Z). The
authors thank the staff of the Department of Bacteriology, Noguchi
Memorial Institute for Medical Research for their support during the
analysis of the samples. The authors also acknowledge the Heads of the
Accra, GIHOC, Madina, Legon and Amasaman abattoirs for their
support during the collection of the samples.
15.
16.
17.
18.
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